Cytotoxic effects of Lavandula angustifolia seed extracts on the viability of Huh-7 and Chang liver cells

Flowering plants are valuable in numerous ways, including food/feed supply for living organ-isms, fuel production, and medicinal uses. Several plant extracts/products are used to treat variety of serious ailments in human and animals. Lavandula angustifolia is a flowering plant that possesses anti-inflammatory and anti-depressive medicinal properties. Cancer is a deadly disorder affecting millions of people globally. It affects several human organs, including liver, stomach, and lungs. Several researchers are doing efforts to eliminate the disease around the globe. In this study, Chang and Huh-7 liver cell lines were utilized as human normal hepatocyte model and innovation to mimic the liver environment. Cytotoxicity of L . angustifolia seed extracts was investigated at two different concentrations (50% and 100%) against Chang and Huh-7 liver cell lines by colorimetric assay which is used to assess cell metabolic activities. The Chang and Huh-7 liver cell lines were treated with L . angustifolia seeds extracts (50% and 100%) and incubated for 24 and 48 hours under standard condi-tions (37˚C, 5% CO 2 ). The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay was employed to quantify cell survival. Seed extracts of L . angustifolia exerted varied cytotoxic effects depending on the concentration and treatment duration. The results indi-cated that L . angustifolia seed extracts with 100% concentration exhibited the highest cytotoxicity against Huh-7 and Chang liver cell lines. In conclusion, L . angustifolia seed extracts exhibited cytotoxic activity which can be enhanced based on the concentration and treatment duration. The findings of the current study are critical for the development of novel herbal-based therapies


Introduction
Humanity has faced several health issues since its beginning. Several diseases pose significant threats to human life. Although medical science has witnessed significant progress in the twenty-first century, there are still several diseases that are incurable. Therefore, cures for these diseases must be investigated. Cancer is a fatal health issue that affects people all over the world. It can arise in almost any tissue/organ of the body when anomalous cells grow uncontrollably, migrate to other organs through metastasizing process and cause death [1]. Cancer is the second leading cause of human mortality globally. World health organization reported 9.6 million (one in six deaths) deaths occurred during 2018 due to cancer [1]. Cancer in most common organs such as liver, stomach, colorectal, prostate, and lung is frequently observed in men, whereas thyroid, cervical, lung, colorectal, and breast cancer are widespread in women [2]. Among different kinds of cancers, incidents of liver cancer are dramatically increasing in males and remain stable in females in the age range of 30-59 years globally [3]. Liver cancer is widely initiated due to hepatitis B and C virus, and fatty liver in younger people. Furthermore, obesity leads to fatty liver disease, which is mounting in both developing and developed nations [4]. It has been suggested that constant rapid increase in liver cancer-mediated deaths is decreasing in women, whereas become stable in men. Therefore, liver cancer is more frequent in young and old populations, particularly in men [5]. However, the role of natural plant-based chemotherapy in liver cancer in vivo and in vitro systems remains poorly understood. Different types of plants have been used in herbalism in various parts of the world in all civilizations at local level. The plant-based products are being suggested by professionals for the treatment of a variety of disorders. Numerous people around the world use herbal medications for a variety of medical needs. Plant species are thought to be a rich source of compounds that can be utilized for making active pharmaceutical or manufactured medications. Plant-based medicines are usually free of adverse effects, which makes them more popular among the public. Plant extracts serve various purposes, including natural coloring, crop pest management, food, cosmetics, and beverage etc. Natural plant species have been used not only as sources of food, but also as remedies for many diseases in the ancient era. Different reports suggested that there are unidentified properties in different parts of plants that can be used in the treatment of certain diseases [6]. The current investigation intended to reveal the potential anticancer and anti-proliferative activities of lavender plant extracts in liver cancer cell line in an in vitro model. The HuH-7 cell line was developed in 1982 from a 57-year-old Japanese male liver tumor patient's hepatocyte cell line. It was then further refined and now being used in vitro to produce hepatitis C virus (HCV) medicines. The unique characters of HuH-7 cell line reveal that it is made up of epithelial-like, tumorigenic cells that stick to the surface of flasks or plates and proliferate as 2D monolayers. According to detailed literature review, this is the first study investigating the impact of lavender seed extracts on liver cancer cells.
Lavender [Lavandula angustifolia Mill (LV) (Lamiales; Lamiaceae)] belongs to the mint family that is native to Mediterranean, Middle Eastern Asian countries, Arabian Peninsula, and Russia, also growing in Europe, United States, and Australia [7]. It has been used in numerous folk medicine traditions for aromatherapy, anxiety, depression, insomnia, headache, food processing, as an antiseptic, massage oil, tea, and source of drugs [8]. In the past decade, considerable scientific literature has reported that the extracts of L. angustifolia possess different pharmacological properties such as anti-neoplastic, antioxidant, anti-inflammatory, antifungal, antiviral, antiseptic, antibacterial, anti-depressive, anticancer, hepatoprotective, and immune-modulatory activities [9][10][11][12][13][14][15][16]. In addition, it is known to be used to cure wide spectrum of respiratory infections, central nervous system cardiovascular disorders, and chronic diseases such as cancer [17]. The L. angustifolia plant extracts are rich in hydrophilic constitutes such as phenolic compound, anthocyanins, phytosterols, tannins, flavones, glycosides, and lipophilic components. Essential oil from the flowers of lavender constituents >300 biologically active chemical compounds, and their stature has been elucidated. Biological and pharmacological properties of essential oils imparted by terpenes and terpenoids [8].
However, clinical research of aromatherapy massage studies does not show a positive outcome in terminal cancer patients. On the other hand, there is enough literature confirmation on the therapeutic benefits of treating cancer patients. Furthermore, ability of L. angustifolia oil to alleviate various associated syndromes in cancer patients have been reported recently. Irrespective of the route of administration, higher concentration of lavender oil could be toxic [18]. Most of the studies have reported that distillation extracts of lavender had cytotoxic and apoptotic effects on various human cancer cells, including prostate cancer (PC-3), lung cancer (A-549), breast cancer (MCF7), and cervical cancer (HeLa). However, effects of L. angustifolia extract on liver cancer cells remain elusive [19]. Keeping in view the advantages of L. angustifolia, present study was conducted to evaluate the anticancer and anti-proliferative effects of lavender extracts from essential oils and their main components on human liver cancer cells. The efficacy of these constitutes was evaluated and compared in both normal and cancer cell culture an in vitro model.

Ethics statement
Seeds of L. angustifolia were collected from northern Saudi Arabia (Al-Khuzama desert, 30.9599˚N, 41.0596˚E). We declare that L. angustifolia seeds were not collected from public parks or protected areas. Moreover, it is not an endangered species in the country.

Plant material
Lavender seeds were collected from northern desert of Saudi Arabia during spring season (April 2020). The collected seeds were dried at 50˚C in an oven for 6 days. The dried seeds (with a moisture content of 8.25%) were cleaned, ground into powder (60-mesh sieve) and stored in plastic bags until use.

Crude extract preparation
Crude extract was prepared following the method of Yagoub et al. [20]. A known weight of lavender seeds was separately soaked in 100% methanol and 50% methanol solution at a solid-toliquid ratio of 1:10 in conical flasks. The flasks containing the mixtures were wrapped with aluminum foil, shaken for 6 h, and contents were filtered using Whatman No. 1 filter paper. The extraction solvents were removed from the filtrates by a vacuum heating at 40˚C using rotary vacuum evaporator (HS-2005S, Hahnshin, S & T Co. Ltd., Korea), and lyophilized. The lyophilized samples were kept in containers and stored at -20˚C until use.

Chemicals and consumables
Cell culture plates and other plasticwares used in the current study were obtained from Tarsons Product Pvt. Ltd. Sigma-Aldrich, USA. The fetal bovine serum (FBS), trypsin-EDTA (0.25%), and Dulbecco's Modified Eagle Medium (DMEM) were obtained from Sigma-Aldrich in the United States. Neutral red dye, MTT, and other chemicals and solvents were of analytical grade and procured from Hi-Media, USA.

Cell culture
Chang and Huh-7 liver cells were kindly provided by Dr. Saud Alarifi, Science College Lab, King Saud University, Saudi Arabia. Huh-7 and Chang liver cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) medium supplemented with 10% fetal bovine serum (FBS), 1% antibiotic/antimycotic solution, and 0.2% NaHCO 3 in a CO 2 incubator at 37˚C in a high humid atmosphere.

Experimental blueprint
The Huh-7 and Chang liver cell lines were exposed to 20-2, 400μg/ml of 100% methanol and 50% methanol extract for 24 h. After extract exposure, cytotoxicity effect of lavender extracts was performed through morphological examination and MTT assay for Huh-7 and Chang liver cell lines.

Cytotoxicity examination of lavender extracts through MTT assay
As explained in Mossman et al. [21], cytotoxic effect of lavender extract was measured through MTT method. Briefly, 10,000 Chang and Huh-7 liver cells were placed in 96 well culture plates and permitted to attach in CO 2 incubator overnight. Afterwards, Chang and Huh-7 liver cell lines were treated with 20-2,400 μg/ml 100% methanol and 50% methanol extracts of lavender for 24 h. After exposure, 5 mg/ml MTT solution (10 μl/well) was added, and incubation continued for additional 4 h. After MTT exposure, culture medium without cells was detached from the wells, and 200 μl of DMSO was supplemented to every well and mixed thoroughly. The absorbance of plates was recorded at 550 nm [21,22].

Statistical analysis
Statistical analysis was executed on SPSS Statistics for Windows, version 21.0 (SPSS Inc., Chicago, IL, USA). The data are mentioned as means ± SE from four independent experiments with three replications each time. The p values (< 0.05) were used for statistically significance between different experimental groups using one-way ANOVA.

Results
Stereological investigation of Huh-7 liver cells' viability exposed with L. angustifolia [50% (LV50%) and 100% (LV100%)] methanol extracts at concentrations from 20 to 2400 μg/ml 24 hours after incubation are shown in Fig 1A and 1B. Cell viability was not decreased by LV50% <200μg/ml, whereas significantly decreased by LV50% concentration >200μg/ml. However, LV100% extracts could not cause cytotoxicity until 2200μg/ml concentration, whereas it significantly decreased by LV100% concentration at 2400μg/ml. Fig 2A and 2B show cell viability of Chang liver cells exposed to different concentrations of L. angustifolia methanol extracts. Cell viability was not affected by LV50%, and LV100% methanol extracts-treated Chang liver cells at concentrations <200μg/ml, whereas significantly decreased by the concentrations >200μg/ml after 24 h incubation compared with control cells. Fig 3A and 3B show cell viability of Huh-7 liver cells exposed to LV50% and LV100% at concentrations from 20 to 2400μg/ml 48 hour after incubation. Cell viability was significantly decreased by LV50% and LV100% methanol extracts at all concentrations 48 h after incubation. The LV100% methanol extracts greatly increased cytotoxicity at >100μg/ml concentrations compared to LV50% 48 hour after incubation. Fig 4A and 4B shows cell viability of Chang liver cells exposed treated with LV50% and LV100% extracts at concentrations ranging from 20 to 2400μg/ml 48 hours after incubation. Cell viability was significantly decreased by LV50% and LV100% methanol extracts at all concentrations 48 hours after incubation. The LV 100% methanol extracts significantly increased cytotoxicity >100μg/ml concentrations compared to LV50% 48 hours after incubation.

Discussion
Researchers around the world are looking at the use of plant-derived medicines as a healthcare supplement [23]. Herbal medicines remain to be standard methods in cancer chemotherapy management. This is due to lacuna of the scientific evidence. However, plant-based compounds represent a massive percentage of pharmaceutical manufacture in the modern world [24]. Identification of biologically targetable compounds that take advantage of divergence between normal and cancerous cells could permit superior specificity for cancer treatment with minimal disadvantage to healthy cells is still the eventual objective in antitumor drug development [25].
Hepatocellular carcinoma (HCC), a type of malignant tumors, is causing high mortality of cancer-related death worldwide. Compared to other forms of cancers, HCC arises from several stages and multifactorial progression. Different risk factors drive the progression of HCC malignancy, and treatments are not effective once the disease is initiated [26,27]. The currently used most important anticancer drugs, including vincristine, vinblastine, and paclitaxel were developed through the investigation of different indigenous preparations [28]. The present study underscores the therapeutic efficacy of L. angustifolia seed extract by indicating its cytotoxic property against Hun7 cells, human hepatocellular carcinoma cells, and Chang liver cells, explaining the cell death mechanism in vitro.
We found that L. angustifolia seed extract has cytotoxic-inducing effects in Huh7 and Chang liver cells in a duration and concentration-dependent mode. The cytotoxic property could be due presence of different pharmacological active principles in LV50% and LV100% PLOS ONE seed extract. Based on the result, we found that 100% methanol extract of L. angustifolia seed extract increased cytotoxicity at 48 h compared to other time. A similar pattern of results we observed that Tridham and its active principles showed cytotoxic property in the human cancer cell lines [29]. Our findings are in line with a prior study that reported a moderate antiproliferative effect of the essential oil of L. angustifolia grown in Brazil against GM07492 human fibroblasts cells [30]. In addition, our MTT results are comparable to the study performed by Najaran and Amiri [31], who found that various extracts and essential oils of L. angustifolia exhibited a promising cytotoxicity against HeLa and MCF-7 cancer cell lines. Another recent work investigated the antiproliferative activity of L. angustifolia essential oils that grown in Tuscany (Italy) showed a notable effect against SHSY5Y neuroblastoma cell line [32], and these variations in results could be attributed to the cell lines used. The outcome of the current investigation discloses that L. angustifolia seed extract induces active growth inhibition in hepatoma cells. Presence of several phytochemicals such as carvacrol, spathulenol, p-cymene-8-ol, caryophyllene oxide, and terpinolene present in L. angustifolia seed extract are responsible for the obtained results. Salleem et al. [33] reported the initiation one of cascade events of cell death known as apoptosis in cancer cell lines.

Conclusions
In conclusion, L. angustifolia seed extract may provide the possibility of cancer prevention and therapy, which is evidenced by the inhibition of cytotoxicity in cancer cell lines. Among the  two tested doses, 100% methanol extract exhibited promising cytotoxic properties. Further, additional investigations are warranted to delineate proper molecular mechanisms involved in the anticancer activity.